Methods for cardiomyocyte survival, proliferation or differentiation by nrg-2

ABSTRACT

The invention features methods of treatment and diagnosis using NRG-2 polypeptides, nucleic acid molecules, and antibodies. The invention also provides novel NRG-2 polypeptides and nucleic acid molecules.

RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 16/116,271, filed Aug. 29, 2018, which is a continuation ofU.S. patent application Ser. No. 14/576,706, filed Dec. 19, 2014 (nowU.S. Pat. No. 10,065,999, issued on Sep. 4, 2018), which is continuationapplication of U.S. patent application Ser. No. 13/372,075 (abandoned),filed Feb. 13, 2012, which is a continuation application of U.S. patentapplication Ser. No. 11/085,812, filed Mar. 21, 2005 (now U.S. Pat. No.8,114,838, issued on Feb. 14, 2012), which is a continuation applicationof U.S. patent application Ser. No. 09/864,675, filed May 23, 2001 (nowU.S. Pat. No. 6,890,751, issued on May 10, 2005), which claims thebenefit of and priority to U.S. Patent Application Ser. No. 60/206,495,filed May 23, 2000, each of which is hereby incorporated by reference inits entirety.

FIELD OF THE INVENTION

The invention relates to neuregulins and methods for their use.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “043509-509C04US_ST25.txt”, whichwas created on Feb. 14, 2019 and is 10.9 KB in size, are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Neuregulins (NRGs) and their receptors constitute a growthfactor-receptor tyrosine kinase system for cell-cell signalling that hasbeen implicated in organogenesis in nerve, muscle, epithelia, and othertissues (Lemke, Mol. Cell. Neurosci. 7: 247-262, 1996; Burden et al.,Neuron 18: 847-855, 1997). The three known NRG genes, NRG-1, NRG-2, andNRG-3, map to distinct chromosomal loci (Pinkas-Kramarski et al., Proc.Natl. Acad. Sci. USA 91: 9387-91, 1994; Carraway et al., Nature 387:512-516, 1997; Chang et al., Nature 387:509-512, 1997; and Zhang et al.,Proc. Natl. Acad. Sci. USA 94: 9562-9567, 1997), and collectively encodea diverse array of NRG proteins. The NRG protein family includes atleast 20 (and perhaps 50 or more) secreted and membrane-bound isoformscontaining epidermal growth factor-like (EGFL), immunoglobulin (Ig), andother recognizable domains.

The most thoroughly studied NRG proteins to date are the gene productsof NRG-1, which include a group of approximately 15 distinctstructurally-related isoforms (Lemke, Mol. Cell. Neurosci. 7: 247-262,1996 and Peles and Yarden, BioEssays 15: 815-824, 1993). Isoforms ofNRG-1 include Neu Differentiation Factor (NDF; Peles et al., Cell 69,205-216, 1992 and Wen et al., Cell 69, 559-572, 1992), Heregulin (HRG;Holmes et al., Science 256: 1205-1210, 1992), Acetylcholine ReceptorInducing Activity (ARIA; Falls et al., Cell 72: 801-815, 1993), and theglial growth factors GGF1, GGF2, and GGF3 (Marchionni et al., Nature362: 312-8, 1993).

The NRG-2 gene was identified by homology cloning (Chang et al., Nature387:509-512, 1997; Carraway et al., Nature 387:512-516, 1997; andHigashiyama et al., J. Biochem. 122: 675-680, 1997) and through genomicapproaches (Busfield et al., Mol. Cell. Biol. 17:4007-4014, 1997). NRG-2isoforms include Neural- and Thymus-Derived Activator of erbB Kinases(NTAK; Genbank Accession No. AB005060), Divergent of Neuregulin (Don-1),and Cerebellum-Derived Growth Factor (CDGF; PCT application WO97/09425). Cells expressing erbB4 or erbB2/erbB4 receptors may show aparticularly robust response to NRG-2 (Pinkas-Kramarski et al., Mol.Cell. Biol. 18: 6090-6101, 1998). The NRG-3 gene product (Zhang et al.,Proc. Natl. Acad. Sci. USA 94: 9562-9567, 1997) is also known to bindand activate erbB4 receptors (Hijazi et al., Int. J. Oncol.13:1061-1067, 1998).

The EGFL domain, present at the core of NRG isoforms, is required forbinding and activating NRG receptors, which belong to the epidermalgrowth factor receptor (EGFR) family, and include EGFR (or erbB1),erbB2, erbB3, and erbB4, also known as HER1 through HER4, respectively,in humans (Meyer et al., Development 124: 3575-3586, 1997; Orr-Urtregeret al., Proc. Natl. Acad. Sci. USA 90: 1867-71, 1993; Marchionni et al.,Nature 362: 312-8, 1993; Chen et al., J. Comp. Neurol. 349: 389-400,1994; Corfas et al., Neuron 14: 103-115, 1995; Meyer et al., Proc. Nal.Acad. Sci. USA 91:1064-1068, 1994; and Pinkas-Kramarski et al., Oncogene15: 2803-2815, 1997). High-affinity binding of the NRGs may be mediatedprincipally via either the erbB3 or erbB4 receptors. Binding of NRGligands leads to dimerization with other erbB subunits andtransactivation by phosphorylation on specific tyrosine residues.

NRG proteins have diverse biological properties, making them potentiallyuseful in the development of novel therapies for a wide range ofdiseases and disorders.

SUMMARY OF THE INVENTION

The invention provides methods of treatment and diagnosis using NRG-2polypeptides, nucleic acid molecules, and antibodies. The invention alsoprovides novel NRG-2 polypeptides and nucleic acid molecules.

In the first aspect, the invention provides a method for increasing themitogenesis, survival, growth, or differentiation of a cell byadministering a NRG-2 polypeptide to the cell in an amount effective forincreasing the mitogenesis, survival, growth, or differentiation of thecell, where the cell expresses an erbB receptor that is selective for aNRG-2 polypeptide. In preferred embodiments of this aspect, the erbBreceptor is an erbB4 homodimer, an erbB2/erbB4 heterodimer, or anerbB1/erbB3 heterodimer. In other preferred embodiments of the firstaspect, the cell is a neuronal cell, a neuronal progenitor cell, aneuronal-associated cell, or a muscle cell. In other preferredembodiments of the first aspect, the neuronal-associated cell is aSchwann cell, an astrocyte, an oligodendrocyte, an O-2A progenitor cell,a glial cell, a microglial cell, an olfactory bulb ensheathing cell, ora sensory organ cell, and the muscle cell is a myoblast, a satellitecell, a myocyte, a skeletal muscle cell, a smooth muscle cell, or acardiac muscle cell.

In a second aspect, the invention provides a method of stimulatingmitogenesis of a glial cell by contacting the glial cell with arecombinant NRG-2 polypeptide. In a preferred embodiment of the secondaspect, the glial cell is an oligodendrocyte, a microglial cell, amyelinating glial cell, an olfactory bulb ensheathing cell, or a glialcell in an adult.

In a third aspect, the invention provides a method for inducingmyelination of a neuronal cell by a glial cell by contacting the glialcell with a NRG-2 polypeptide, such that the contacting is sufficient toinduce myelination of the neuronal cell by the glial cell.

In a fourth aspect, the invention provides a method of increasing thecardiomyocyte survival, cardiomyocyte proliferation, cardiomyocytegrowth, or cardiomyocyte differentiation, in a mammal in need thereof,by administering a NRG-2 polypeptide to the mammal in an amounteffective for increasing the cardiomyocyte survival, cardiomyocyteproliferation, cardiomyocyte growth, or cardiomyocyte differentiation.In preferred embodiments of the fourth aspect, the mammal is a human. Inother preferred embodiments of the fourth aspect, the mammal has apathophysiological condition that affects cardiac muscle, for example,cardiomyopathy (e.g., a degenerative congenital disease), cardiactrauma, heart failure, or ischemic damage, or the mammal has apathophysiological condition that affects smooth muscle, for example,atherosclerosis, vascular lesion, vascular hypertension, or degenerativecongenital vascular disease. In another preferred embodiment of thefourth aspect the mammal is a patient with myasthenia gravis.

In a fifth aspect, the invention provides a method of affecting cellularcommunication between a neuronal-associated cell and a neuronal cell ina mammal by administering a NRG-2 polypeptide to the mammal, such thatthe neuregulin interacts with the neuronal-associated cell, resulting inthe production of at least one neurotrophic agent by theneuronal-associated cell, and the neurotrophic agent or agents affectthe mitogenesis, survival, growth, differentiation, or neurite outgrowthof the neuronal cell. In a preferred embodiment of the fifth aspect, themammal is a human. In other preferred embodiments of the first aspect,the neuronal-associated cell is a Schwann cell, an astrocyte, anoligodendrocyte, an O-2A progenitor cell, a glial cell, an olfactorybulb ensheathing cell, a microglial cell, a sensory organ cell, or amuscle cell (e.g., a skeletal muscle cell, a smooth muscle cell, or acardiac muscle cell). In other preferred embodiments of the fifthaspect, the cellular communication is affected in the central nervoussystem or the peripheral nervous system of a mammal. In other preferredembodiments of the fifth aspect, the administering includesadministering a purified NRG-2 polypeptide-producing cell.

In a sixth aspect, the invention provides a method for the treatment orprophylaxis of a pathophysiological condition of the nervous system in amammal, by administering a therapeutically-effective amount of arecombinant NRG-2 polypeptide to the mammal. In preferred embodiments ofthe sixth aspect, the pathophysiological condition is a condition of theperipheral nervous system or the central nervous system; thepathophysiological condition is demyelination of nerve cells, damage ofSchwann cells, loss of Schwann cells, or a neurodegenerative disorder;the pathophysiological condition is a peripheral neuropathy (e.g., asensory nerve fiber neuropathy, a motor fiber neuropathy, or both); orthe pathophysiological condition is multiple sclerosis, amyotrophiclateral sclerosis, spinal muscular atrophy, nerve injury, Alzheimer'sDisease, Parkinson's Disease, cerebellar ataxia, or spinal cord injury.In another preferred embodiment of the sixth aspect, the treatment orprophylaxis requires neural regeneration or neural repair. In anotherpreferred embodiment of the sixth aspect, the NRG-2 polypeptideinteracts with neuronal-associated cells, resulting in production of atleast one neurotrophic agent by the neuronal-associated cells, and theneurotrophic agent or agents affect the mitotic activity, survival,differentiation or neurite outgrowth of neuronal cells. In anotherpreferred embodiment of the sixth aspect, the administering issufficient to induce myelination of a neuronal cell by a glial cell(e.g., a Schwann cell or an oligodendrocyte). In another preferredembodiment of the sixth aspect, the administering includes administeringa purified NRG-2 polypeptide-producing cell to the mammal. The NRG-2polypeptide-producing cell of the invention may contain a recombinantDNA sequence, where the DNA sequence includes a NRG-2polypeptide-encoding sequence, and where the NRG-2 polypeptide-encodingDNA sequence is operably-linked to a promoter.

In a seventh aspect, the invention provides a method for the treatmentof a tumor (e.g., a glial tumor) by inhibiting the proliferation of atumor cell by administering an effective amount of an antibody, thatinhibits binding of a NRG-2 polypeptide to a receptor present on thesurface of the tumor cell, to a subject in need of such treatment. In apreferred embodiment of the seventh aspect, the tumor cell expresses anerbB receptor that is selective for a NRG-2 polypeptide.

In an eighth aspect, the invention provides a method for the treatmentof neurofibromatosis by inhibiting glial cell mitogenesis byadministering an effective amount of an antibody, that inhibits bindingof a NRG-2 polypeptide to a receptor present on the surface of a glialtumor cell in an individual with neurofibromatosis, to a subject in needof such treatment.

In a ninth aspect, the invention provides a method for inhibitingproliferation of a cell by contacting the cell with an effective amountof an antibody that inhibits binding of a NRG-2 polypeptide to areceptor present on the surface of the cell.

In a tenth aspect, the invention provides a method for stimulatingproliferation of a cell by administering a NRG-2 polypeptide to thecell.

In preferred embodiments of the ninth and tenth aspects, the cell is aneuronal cell, a neuronal-associated cell, or a muscle cell.

The NRG-2 polypeptide of any of the above aspects or embodiments of theinvention may include, or consist of, the amino acid sequences set forthin SEQ ID NOs: 2 or 4, or be encoded by the nucleic acid sequences setforth in SEQ ID NOs: 1 or 3.

In an eleventh and a twelfth aspect, the invention provides asubstantially pure NRG-2 polypeptide including, or consisting of, theamino acid sequences set forth in SEQ ID NOs: 2 or 4. In a thirteenthaspect, the invention provides a substantially pure nucleic acidmolecule including a sequence encoding a polypeptide including the aminoacid sequences set forth in SEQ ID NOs: 2 or 4. In preferredembodiments, the invention provides a vector (e.g., a gene therapyvector) including the nucleic acid molecule of the thirteenth aspect,operably linked to a promoter; a cell containing a gene therapy vectorthat contains the nucleic acid molecule of the thirteenth aspect; and anon-human transgenic animal containing the nucleic acid molecule of thethirteenth aspect.

In fourteenth and fifteenth aspects, the invention provides asubstantially pure nucleic acid molecule including, or consisting of, anucleic acid sequence that is substantially identical to the nucleicacid sequences set forth in SEQ ID NOs: 1 or 3. In a sixteenth aspect,the invention provides a nucleic acid molecule including a sequence thatis antisense to the coding strand sequence of the nucleic acid sequenceset forth in SEQ ID NOs: 1 or 3, or a fragment thereof.

In a seventeenth aspect, the invention provides a non-human animalhaving a knockout mutation in one or both alleles encoding the NRG-2polypeptide including the amino acid sequence set forth in SEQ ID NOs: 2or 4. In a preferred embodiment of the seventeenth aspect, the inventionprovides a cell from the non-human animal of the seventeenth aspect.

In an eighteenth aspect, the invention provides an antibody thatspecifically binds to a NRG-2 polypeptide that includes the amino acidsequences set forth in SEQ ID NOs: 2 or 4. In a preferred embodiment ofthe eighteenth aspect, the invention provides a method of detecting thepresence of a NRG-2 polypeptide in a sample by contacting the samplewith the antibody of the eighteenth aspect, and assaying for binding ofthe antibody to the polypeptide. In a preferred embodiment of theeighteenth aspect, the invention provides a kit for the analysis of aNRG-2 polypeptide of a test subject, where the kit includes the antibodyof the eighteenth aspect.

In a nineteenth aspect, the invention provides a method of diagnosing anincreased likelihood of developing a NRG-2-related disease or conditionin a test subject (e.g., a human) by analyzing nucleic acid molecules ofthe test subject, to determine whether the test subject contains amutation in NRG-2 gene that encodes a NRG-2 polypeptide including theamino acid sequence set forth in SEQ ID NOs: 2 or 4, where the presenceof the mutation is an indication that the test subject has an increasedlikelihood of developing a NRG-2-related disease. By “neuregulin” or“NRG” is meant a polypeptide that is encoded by a NRG-1, NRG-2, or NRG-3gene or nucleic acid molecule (e.g., a cDNA), and binds to and activatesan erbB receptor or combinations thereof. Generally, a neuregulinpossesses recognizable domains, such as an epidermal growth factor-like(EGFL) domain, that binds to and activates an erbB receptor orcombinations thereof, and an immunoglobulin (Ig) domain. An EGFL domainbears structural similarity to the EGF receptor-binding domain asdisclosed in Holmes et al. (Science 256:1205-1210, 1992), U.S. Pat. Nos.5,530,109, 5,716,930, U.S. Ser. No. 08/461,097, Hijazi et al. (Int. J.Oncol. 13:1061-1067, 1998), Chang et al. (Nature 387:509-512, 1997),Carraway et al. (Nature 87:512-516, 1997), Higashiyama et al. (J.Biochem. 122: 675-680, 1997), and PCT publication WO 97/09425.

By “neuregulin 2” or “NRG-2” is meant a polypeptide encoded by a NRG-2gene, or a NRG-2 nucleic acid molecule, and is described in thespecification herein, and in, for example, Carraway et al., Nature 387:512-516, 1997; Chang et al., Nature 387: 509-511, 1997; Higashiyama etal., J. Biochem. 122: 675-680, 1997; and Busfield et al., Mol. Cell.Biol. 17:4007-4014, 1997. NRG-2 isoforms include Neural- andThymus-Derived Activator of ErbB Kinases (NTAK; Genbank Accession No.AB005060; Higashiyama et al., J. Biochem. 122: 675-680, 1997), Divergentof Neuregulin (don-1; PCT publication WO 98/07736), andCerebellum-Derived Growth Factor (CDGF; PCT publication WO 97/09425;U.S. Pat. No. 5,912,326), incorporated by reference herein, and theNRG-2 molecules described herein. Generally, the erbB1 receptor (EGFR)is a preferred dimerization partner for a NRG-2 polypeptide. Preferredreceptor combinations for NRG-2 polypeptides are erbB4 homodimers,erbB2/erbB4 heterodimers, or erbB1/erbB3 heterodimers. A CDGF, don-1, orNTAK polypeptide or nucleic acid molecule, as set forth in the aminoacid and nucleic acid sequences disclosed in Higashiyama et al., J.Biochem. 122: 675-680, 1997, WO 98/07736, WO 97/09425, and U.S. Pat. No.5,912,326, may be specifically excluded from certain aspects of theinvention. For example, one or more of a CDGF, don-1, or NTAKpolypeptide or nucleic acid molecule, may be excluded from the methodsfor increasing the mitogenesis, survival, growth, or differentiation ofa cell; for increasing the cardiomyocyte survival, cardiomyocyteproliferation, cardiomyocyte hypertrophy, or cardiomyocytedifferentiation in a mammal; for affecting cellular communicationbetween a neuronal-associated cell and a neuronal cell; for stimulatingmitogenesis of a glial cell; for inducing myelination of a neuronal cellby a glial cell; for the treatment or prophylaxis of apathophysiological condition of the nervous system in a mammal; for thetreatment of a tumor; for the treatment of neurofibromatosis; forinhibiting proliferation of a cell; or for stimulating proliferation ofa cell.

By “erbB receptor” is meant erbB1 (EGFR), erB2, erbB3, and erbB4 (alsoHER-1, HER-2, HER-3, and HER-4 of human) existing as monomeric ormultimeric (e.g., homodimeric or heterodimeric) cell surface receptortyrosine kinases that bind to and/or are activated by one or moreneuregulins (Meyer et al., Development 124: 3575-3586, 1997;Orr-Urtreger et al., Proc. Natl. Acad. Sci. USA 90: 1867-71, 1993;Marchionni et al., Nature 362: 312-8, 1993; Chen et al., J. Comp.Neurol. 349: 389-400, 1994; Corfas et al., Neuron 14: 103-115, 1995;Meyer et al., Proc. Natl. Acad. Sci. USA 91:1064-1068, 1994; andPinkas-Kramarski et al., Oncogene 15: 2803-2815, 1997). Preferably, theerbB receptors are erbB4 homodimers, erbB2/erbB4 heterodimers,erbB1/erbB3 heterodimers, or any receptor combination that is selectivefor a NRG-2 polypeptide over a NRG-1 polypeptide or a NRG-3 polypeptide.

By “selective” is meant the preferential binding of an erbB receptor orcombination thereof to a NRG-2 polypeptide over a NRG-1 or a NRG-3polypeptide. More specifically, preferential binding is defined as anincrease in the affinity of an erbB receptor to a NRG-2 polypeptide ofat least 1.5 fold, more preferably at least 2 fold, relative to theaffinity of an erbB receptor to a NRG-1 or NRG-3 polypeptide.

By “neuronal cell” is meant a neuron, nerve cell, neurocyte, or neuronalprogenitor cell. A neuronal cell is the morphological and functionalunit of the central nervous system and the peripheral nervous system,and includes cholinergic neurons and non-cholinergic neurons.

By “neuronal-associated cell” is meant any non-neuronal cell that iscapable of affecting the function of a neuron, or whose function can beaffected by a neuron. A neuronal-associated cell includes, but is notlimited to, a muscle cell or a nervous system support cell, including aSchwann cell, an astrocyte, an oligodendrocyte, an O-2A progenitor cell,a glial cell (e.g., a radial glial cell or a Bergmann glial cell), amicroglial cell, an olfactory bulb ensheathing cell, or a sensory organcell (e.g., a retinal cell).

By “muscle cell” is meant any cell that contributes to muscle tissue.Muscle tissue is a primary tissue, consisting mainly of specializedcontractile cells, and is generally classified as skeletal muscle,cardiac muscle, or smooth muscle. Myoblasts, satellite cells, myotubes,myocytes (e.g., cardiomyocytes), and myofibril tissues are all includedin the term “muscle cells,” and may all be treated according to themethods of the invention. Muscle cell effects may be induced withinskeletal, cardiac, and smooth muscle.

By “neurotrophic agent” or “neurotrophic factor” is meant a substancethat elicits a trophic effect in one or more neuronal cells. Theseeffects include, but are not limited to, survival, mitosis, anddifferentiation. Neurotrophic agents include, but are not limited to,neurotrophins, nerve growth factor, ciliary neurotrophic factor, andbrain-derived neurotrophic factor.

By “affecting” is meant the induction of a quantitative change in theresponse of a target cell, as a result of an interaction with a NRG-2polypeptide or nucleic acid molecule.

By “cellular communication” is meant the synthesis of a substance (e.g.,a neurotrophic agent) in a first cell type (e.g., a neuronal-associatedcell) and the interaction of that substance with a second cell type(e.g., a neuronal cell), such that the substance elicits a change in thefirst or second cell type. Cellular communication includes, but is notlimited to, secretion of the substance from a cell. Cellularcommunication can occur reciprocally or non-reciprocally with one ormore cell types.

By “mitogenesis” is meant any cell division that results in theproduction of new cells in the patient. More specifically, mitogenesisin vitro is defined as an increase in mitotic index, relative tountreated cells, of 50%, more preferably 100%, and most preferably 300%,when the cells are exposed to labeling agent for a time equivalent totwo doubling times. The mitotic index is the fraction of cells inculture that have labeled nuclei when grown in the presence of a tracerthat only incorporates during S phase (e.g., BrdU), and the doublingtime is defined as the average time required for the number of cells inthe culture to increase by a factor of two. By “inhibiting mitogenesis”is meant a decrease in the mitotic index, relative to untreated cells,of 50%, more preferably 100%, and most preferably 300%, when the cellsare exposed to labeling agent for a time equivalent to two doublingtimes. Inhibiting mitogenesis also means a cessation of any increase inthe mitotic index, relative to control cells.

An effect on mitogenesis in vivo is defined as an increase in cellactivation as measured by the appearance of labeled cells in the tissueof a mammal exposed to a tracer that only incorporates during S phase(e.g., BrdU). An useful therapeutic is defined in vivo as a compoundthat increases cell activation relative to a control mammal by at least10%, more preferably by at least 50%, and most preferably by more than200% when the mammal is exposed to labeling agent for a period ofgreater than 15 minutes and tissues are assayed between 10 hours and 24hours after administration of the mitogen at the therapeutic dose. Forexample, in muscle cells, satellite cell activation in vivo may bedetected by monitoring BrdU incorporation. Alternatively, satellite cellactivation in vivo may be detected by the appearance of the intermediatefilament vimentin by immunological or RNA analysis methods. Whenvimentin is assayed, the useful mitogen is defined as one which causesexpression of detectable levels of vimentin in the muscle tissue whenthe therapeutically useful dosage is provided. Mitogenesis may beinduced in, for example, muscle cells of skeletal, cardiac, and smoothmuscle, and in glial cells.

By “survival” is meant any process by which a cell avoids death. Theterm survival as used herein also refers to the prevention of cell lossas evidenced by necrosis, apoptosis, or the prevention of othermechanisms of cell loss. Increasing survival as used herein indicates adecrease in the rate of cell death by at least 10%, more preferably byat least 50%, and most preferably by at least 100% relative to anuntreated control. The rate of survival may be measured by countingcells capable of being stained with a dye specific for dead cells (e.g.,propidium iodide) in culture. The rate of survival may be measured bycounting cells stainable with a dye specific for dead cells (such aspropidium iodide) in culture when the cells are 8 dayspost-differentiation (i.e., 8 days after the medium is changed from 20%to 0.5% serum).

By “growth” is meant the increase in size or number of a cell typerelative to a control cell. The therapeutic usefulness of growthincreases the size or number of a cell in diseased tissue by at least10% or more, more preferably by 50% or more, and most preferably by morethan 100% relative to the equivalent tissue in a similarly treatedcontrol animal. Growth can be measured by, for example, an increase innet weight, protein content, or cell diameter. Muscle growth may occurby the increase in the fiber size and/or by increasing the number offibers.

By “differentiation” is meant a morphological and/or chemical changethat results in the generation of a different cell type or state ofspecialization. The differentiation of cells as used herein refers to acellular development program that specifies one or more components of acell type. The therapeutic usefulness of differentiation increases thequantity of any component of a cell in diseased tissue by at least 10%or more, more preferably by 50% or more, and most preferably by morethan 100% relative to the equivalent tissue in a similarly treatedcontrol animal.

By “proliferation” is meant the growth or reproduction of similar cells.By “inhibiting proliferation” is meant the decrease in the number ofsimilar cells by at least 10%, more preferably by at least 20%, and mostpreferably by at least 50%. By “stimulating proliferation” is meant anincrease in the number of similar cells by at least 10%, more preferablyby at least 20%, and most preferably by at least 50%.

By “inducing myelination” is meant the acquisition, development, orformation of myelin sheath around a nerve fiber. The useful therapeuticfor inducing myelination confers an increase in the density of a myelinsheath by at least 10%, more preferably by at least 20%, and mostpreferably by at least 50%, relative to a control nerve fiber. By“demyelination” is meant the loss of the myelin sheath around a nervefiber.

By “interacts” is meant contact of a NRG-2 polypeptide with a receptoror other molecule on a target cell.

By “pathophysiological condition” is meant a disturbance of functionand/or structure of a living organism, resulting from an externalsource, a genetic predisposition, a physical or chemical trauma, or acombination of the above, including, but not limited to, any mammaliandisease.

By “neuropathy” is meant any disorder affecting the nervous system. Aneuropathy may be, for example, a peripheral neuropathy, such a sensorynerve fiber neuropathy or motor fiber neuropathy.

By “cardiomyopathy” is meant a disease that affects the heart muscle.Cardiomyopathy may be primary, i.e., mainly affecting cardiac muscle, orsecondary, i.e., affecting cardiac muscle secondary to a systemicdisease, infection, or metabolic disease.

By “ischemic damage” is meant damage resulting from decreased bloodcirculation to cardiac muscle.

By “degenerative congenital disease” is meant a disease that exists atbirth, which may be hereditary or due to an influence occurring duringgestation, that results in a pathological change in cells or tissues.

By “treatment” is meant the medical management of a patient with theintent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement or associated with the cure of a disease, pathologicalcondition, or disorder, and also includes causal treatment, that is,treatment directed toward removal of the cause of the associateddisease, pathological condition, or disorder. In addition, this termincludes palliative treatment, that is, treatment designed for therelief of symptoms rather than the curing of the disease, pathologicalcondition, or disorder; preventative treatment, that is, treatmentdirected to minimizing or partially or completely inhibiting thedevelopment of the associated disease, pathological condition, ordisorder; and supportive treatment, that is, treatment employed tosupplement another specific therapy directed toward the improvement ofthe associated disease, pathological condition, or disorder. The phrase“treatment” also includes symptomatic treatment, that is, treatmentdirected toward constitutional symptoms of the associated disease,pathological condition, or disorder.

By “therapeutically-effective amount” is meant an amount of a NRG-2polypeptide or nucleic acid molecule sufficient to produce a healing,curative, stabilizing, or ameliorative effect in the treatment of adisorder.

By “neurodegenerative disorder” is meant any pathophysiologicalcondition that is characterized by the degeneration of neuronal cells orneuronal-associated cells. The degeneration may include, for example, adecrease in cell number or size, an increase in cell apoptosis or death,or a decrease in cell growth, survival or differentiation.

By “neural regeneration or neural repair” is meant the treatment of apathophysiological condition by, for example, an increase in neuronalcell or neuronal-associated cell number or size, a decrease in neuronalcell or neuronal-associated cell apoptosis or death, or an increase inneuronal cell or neuronal-associated cell growth, survival ordifferentiation.

By “inhibits binding” is meant preventing or reducing the binding of aNRG-2 polypeptide to a receptor. The binding is preferably reduced by atleast 10%, more preferably by at least 50%, and most preferably by atleast 100% relative to a control sample.

By “polypeptide” or “polypeptide fragment” is meant a chain of two ormore amino acids, regardless of any post-translational modification(e.g., glycosylation or phosphorylation), constituting all or part of anaturally or non-naturally occurring polypeptide. By “post-translationalmodification” is meant any change to a polypeptide or polypeptidefragment during or after synthesis. Post-translational modifications canbe produced naturally (such as during synthesis within a cell) orgenerated artificially (such as by recombinant or chemical means). A“protein” can be made up of one or more polypeptides.

The term “identity” is used herein to describe the relationship of thesequence of a particular nucleic acid molecule or polypeptide to thesequence of a reference molecule of the same type. For example, if apolypeptide or nucleic acid molecule has the same amino acid ornucleotide residue at a given position, compared to a reference moleculeto which it is aligned, there is said to be “identity” at that position.The level of sequence identity of a nucleic acid molecule or apolypeptide to a reference molecule is typically measured using sequenceanalysis software with the default parameters specified therein, such asthe introduction of gaps to achieve an optimal alignment (e.g., SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705, BLAST, or PILEUP/PRETTYBOX programs). These software programsmatch identical or similar sequences by assigning degrees of identity tovarious substitutions, deletions, or other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine, valine, isoleucine, and leucine; asparticacid, glutamic acid, asparagine, and glutamine; serine and threonine;lysine and arginine; and phenylalanine and tyrosine.

A nucleic acid molecule or polypeptide is said to be “substantiallyidentical” to a reference molecule if it exhibits, over its entirelength, at least 90%, preferably at least 95%, more preferably at least97%, and most preferably 99% identity to the sequence of the referencemolecule. For polypeptides, the length of comparison sequences is atleast 16 amino acids, preferably at least 20 amino acids, morepreferably at least 25 amino acids, and most preferably at least 35amino acids. For nucleic acid molecules, the length of comparisonsequences is at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably at least 110 nucleotides.

A nucleic acid molecule or polypeptide is “analyzed” or subject to“analysis” if a test procedure is carried out on it that allows thedetermination of its biological activity or whether it is wild type ormutated. For example, one can analyze the genes of an animal (e.g., ahuman) by amplifying genomic DNA of the animal using the polymerasechain reaction, and then determining whether the amplified DNA containsa mutation, e.g., by nucleotide sequence or restriction fragmentanalysis.

By a “substantially pure polypeptide” is meant a polypeptide (or afragment thereof) that has been separated from proteins and organicmolecules that naturally accompany it. Typically, a polypeptide issubstantially pure when it is at least 60%, by weight, free from theproteins and naturally-occurring organic molecules with which it isnaturally associated. Preferably, the polypeptide is a NRG-2 polypeptidethat is at least 75%, more preferably at least 90%, and most preferablyat least 99%, by weight, pure. A substantially pure NRG-2 polypeptidecan be obtained, for example, by extraction from a natural source (e.g.,cerebellum), by expression of a recombinant nucleic acid moleculeencoding a NRG-2 polypeptide, or by chemical synthesis. Purity can bemeasured by any appropriate method, e.g., by column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

A polypeptide is substantially free of naturally associated componentswhen it is separated from those proteins and organic molecules thataccompany it in its natural state. Thus, a protein that is chemicallysynthesized or produced in a cellular system different from the cell inwhich it is naturally produced is substantially free from its naturallyassociated components. Accordingly, substantially pure polypeptides notonly include those derived from eukaryotic organisms, but also thosesynthesized in E. coli or other prokaryotes.

An antibody is said to “specifically bind” to a polypeptide if itrecognizes and binds to the polypeptide (e.g., a NRG-2 polypeptide), butdoes not substantially recognize and bind to other molecules (e.g.,non-NRG-2 related polypeptides) in a sample, e.g., a biological sample,that naturally includes the polypeptide.

By a “transgene” is meant a DNA molecule that is inserted by artificeinto a cell (e.g., the nuclear genome of a cell), and is incorporatedinto the genome of an organism that develops from the cell. Such atransgene can be partly or entirely heterologous (i.e., foreign) to thetransgenic organism, or can be a gene that is homologous to anendogenous gene of the organism. An organism or animal (e.g., a mammal,such as a mouse, rat, or goat) can be said to be “transgenic” if itdeveloped from a cell that had a transgene inserted into it by artifice.

By a “knockout mutation” is meant an artificially-induced alteration ina nucleic acid molecule (created by recombinant DNA technology ordeliberate exposure to a mutagen) that reduces the biological activityof the polypeptide normally encoded therefrom by at least 80% relativeto the unmutated gene. The mutation can be, without limitation, aninsertion, deletion, frameshift mutation, or a missense mutation. A“knockout animal” is preferably a mammal, and more preferably a mouse,containing a knockout mutation, as defined above. By “vector” is meant agenetically engineered plasmid or virus, derived from, for example, abacteriophage, adenovirus, retrovirus, poxvirus, herpesvirus, orartificial chromosome, that is used to transfer a polypeptide (e.g., aNRG-2 polypeptide) coding sequence, operably linked to a promoter, intoa host cell, such that the encoded peptide or polypeptide is expressedwithin the host cell.

By “promoter” is meant a minimal sequence sufficient to direct orcontrol transcription. Also included are those promoter elements whichare sufficient to render promoter-dependent gene expression controllablefor cell type or physiological status (e.g., hypoxic versus normoxicconditions), or inducible by external signals or agents; such elementsmay be located in the 5′ or 3′ or internal regions of the native gene.

By “operably linked” is meant that a nucleic acid encoding a polypeptide(e.g., a cDNA) and one or more regulatory sequences are connected insuch a way as to permit gene expression when the appropriate molecules(e.g., transcriptional activator proteins) are bound to the regulatorysequences.

By “NRG-2 polypeptide producing cell” is meant a cell (or a descendentof a cell) into which a DNA molecule encoding a NRG-2 polypeptide hasbeen introduced, by means of recombinant DNA techniques or known genetherapy techniques.

The invention provides several advantages. For example, it providesmethods and reagents that can be used in the diagnosis and treatment ofdiseases that are sensitive to the bioactivities of NRG-2 polypeptides.Other features and advantages of the invention will be apparent from thedetailed description of the invention, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of NRG-2 gene products and human cDNAclones.

FIG. 2 shows a schematic diagram of the mammalian expression vectorpRc/CMV2.

FIG. 3A shows a western blot analysis of conditioned media (cm) and celllysates (cells) from mock- (vector) or rhNRG-2α-transfected CHO/S cells.

FIG. 3B shows a western blot analysis of conditioned media frommock-(vec), rhNRG-2α (α), or rhNRG-2β-transfected (β) CHO/S cells.

FIG. 3C shows a western blot analysis of conditioned media from CHOcells expressing rat NRG-2β.

FIGS. 4A-B show western blot analyses of receptor phosphorylation ontyrosine residues in cells responsive to treatment with NRG-2polypeptides.

FIG. 5 shows a western blot analysis of the expression of recombinanthuman NRG-2α and NRG-2β.

FIG. 6 shows a western blot analysis of stable transfectants used formethotrexate selection.

FIG. 7A shows a Coomassie stained polyacrylamide gel of purifiedrhNRG-2β.

FIG. 7B shows a scan of the gel of FIG. 7A.

FIG. 8 shows a gold stained polyacrylamide gel of total protein andpurified rhNRG-2β.

FIG. 9 shows a western blot analysis of receptor phosphorylation oftyrosine residues in cells responsive to HPLC purified rhNRG-2β.

FIG. 10A shows a photograph of midbrain dopaminergic neurons pretreatedwith rhNRG-2β and challenged with 6-OHDA.

FIG. 10B shows a photograph of untreated control midbrain dopaminergicneurons challenged with 6-OHDA.

FIG. 11A shows a photograph indicating BrdU incorporation in neuralcells, including neural progenitor cells, cultured with rhNRG-2β.

FIG. 11B shows a bar graph indicating the effect of rhNRG-2β on BrdUincorporation.

FIG. 12A shows a photograph of cerebellar granule neurons migrating on aglial cell process.

FIG. 12B shows a bar graph indicating the effect of NRG-2 on neuronalmigration.

FIG. 13 shows a western blot analysis of the activation of p42/44MAPk(Erk) and Akt by NRG-2 proteins.

FIG. 14 shows a line graph of ³H-Leucine uptake into neonatal ratventricular myocytes treated with NRG proteins.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides NRG-2 polypeptides and nucleic acid molecules,antibodies that bind these NRG-2 polypeptides, and therapeutic anddiagnostic methods employing NRG-2 polypeptides and nucleic acidmolecules.

Bioassays

NRG-2 ligands and erbB receptors are expressed in the nervous system, inneural precursors and neurons of brain, spinal cord and retina; skeletaland cardiac muscle; lung; thymus, kidney; adrenal glands; skin; breastepithelia; and other organs during embryonic development and in adulttissues. Major sites of NRG-2 expression include the cerebellum(Purkinje and granule cells), olfactory bulb, dentate gyrus, pyramidalcells of the occipital cortex, lung, and thymus. The expression patternsof NRG-2 receptors in specific cells and tissues are used to identifycellular targets of NRG-2 actions, and to identify bioactivities thatare relevant to specific NRG-2-related diseases, such as demyelinatingdisorders of the peripheral and central nervous systems; neuropathies;neurodegenerative disorders; cardiomyopathies; loss of hearing, balanceor vision; pain; neurotrauma; cancer; sensorineural hearing loss orsensorineural balance loss from viral infection, aging, or antibiotics(e.g. aminoglycosides); retinopathy (e.g., hypertensive, diabetic,occlusive, macular degeneration, retinitis pigmentosa, optic neuropathy,injury); Guillaime Barre disease; stroke; or brain or spinal cordinjury. Many of the NRG-2-responsive cell types in embryonic, neonatal,and adult tissues express the receptor combinations of erbB2/erbB3,erbB2/erbB4, or erbB4 alone. For example, peripheral nervous system(PNS) and central nervous system (CNS) glial cell types express erbB2;Schwann cells also express erbB3. In the CNS, erbB4 and erbB3 have beenobserved on various glial cell types, including astrocytes,oligodendrocyte progenitors, radial glia in the developing cortex, andBergmann glia in the cerebellum. The erbB2/erbB4 combination is found inventricular cardiomyocytes.

Therapeutic and diagnostic utilities for NRG-2 polypeptides areidentified by, for example, conducting bioassays in vitro. Culturesystems that reflect NRG-2 expression patterns, along with thedistribution of particular receptors, such as erbB2/erbB4 or erbB4alone, which are examples of erbB receptor combinations that may show apreference for NRG-2 over NRG-1, are selected. For example, NRG-2bioactivities are evaluated using CNS glia, such as oligodendrocytes andolfactory bulb ensheathing cells, mid-brain dopaminergic neurons,cerebellar granule neurons, and cardiomyocytes. These cell populationsexpress NRG receptors, and respond to treatment with one or moreisoforms of NRG-1 in a variety of quantitative bioassays. The activitiesof NRG-2 (e.g. rhNRG-2α, rhNRG-2β) and NRG-1 (e.g., rhGGF2) isoforms arecompared, using sister cultures, in various dose-response assays,including but not limited to, stimulation of proliferation, survival,differentiation, migration, and morphological changes. The relativepotencies of the NRG-2 and NRG-1 isoforms are determined on the basisof, for example, protein concentration.

Diagnostic Methods Employing NRG-2 Nucleic Acid Molecules, Polypeptides,and Antibodies

NRG-2 nucleic acid molecules, polypeptides, and antibodies are used inmethods to diagnose or monitor a variety of diseases and conditions,including those involving mutations in, or inappropriate expression of,NRG-2 genes. NRG-2 expression has been documented in a variety oftissues, as discussed above. Thus, detection of abnormalities in NRG-2genes or their expression are used in methods to diagnose, or to monitortreatment or development of diseases of these tissues.

The diagnostic methods of the invention are used, for example, withpatients that have a cardiovascular or a neurological disease, in aneffort to determine its etiology, and thus, to facilitate selection ofan appropriate course of treatment. The diagnostic methods are also usedwith patients that have not yet developed a cardiovascular orneurological disease, but who may be at risk of developing such adisease, or with patients that are at an early stage of developing sucha disease. Many cardiovascular and neurological diseases occur duringdevelopment, and thus, the diagnostic methods of the invention are alsocarried out on a fetus or embryo during development. Also, thediagnostic methods of the invention are used in prenatal geneticscreening, for example, to identify parents who may be carriers of arecessive NRG-2 mutation.

NRG-2 abnormalities that are detected using the diagnostic methods ofthe invention include those characterized by, for example, (i) abnormalNRG-2 polypeptides, (ii) NRG-2 genes containing mutations that result inthe production of such polypeptides, and (iii) NRG-2 mutations thatresult in production of abnormal amounts of NRG-2.

Levels of NRG-2 expression in a patient sample are determined by usingany of a number of standard techniques that are well known in the art.For example, NRG-2 expression in a biological sample (e.g., a blood ortissue sample, or amniotic fluid) from a patient is monitored bystandard northern blot analysis or by quantitative PCR (see, e.g.,Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, N.Y., 1998; PCR Technology: Principles and Applicationsfor DNA Amplification, H. A. Ehrlich, Ed., Stockton Press, NY; Yap etal. Nucl. Acids. Res. 19:4294, 1991).

Therapeutic Methods Employing NRG-2 Nucleic Acid Molecules,Polypeptides, and Antibodies

The invention includes methods of treating or preventing NRG-2-relateddiseases. Therapies are designed to circumvent or overcome a NRG-2 genedefect, or inadequate or excessive NRG-2 gene expression, and thusmodulate and possibly alleviate conditions involving defects in NRG-2genes or proteins. In considering various therapies, it is understoodthat such therapies are, preferably, targeted to the affected orpotentially affected organs, for example, the heart or the nervoussystem. Reagents that are used to modulate NRG-2 biological activity caninclude, without limitation, full length NRG-2 polypeptides; NRG-2 cDNA,mRNA, or antisense RNA; NRG-2 antibodies; and any compound thatmodulates NRG-2 biological activity, expression, or stability.

Treatment or prevention of diseases resulting from a mutated NRG-2 geneis accomplished, for example, by replacing a mutant NRG-2 gene with anormal NRG-2 gene, administering a normal NRG-2 gene, modulating thefunction of a mutant NRG-2 protein, delivering normal NRG-2 protein tothe appropriate cells, or altering the levels of normal or mutant NRG-2protein. It is also possible to correct a NRG-2 defect to modify thephysiological pathway (e.g. a signal transduction pathway) in which theNRG-2 protein participates.

Gene transfer is achieved using viral vectors, as well as non-viralmeans involving transfection in vitro by means of any standardtechnique, including but not limited to, calcium phosphate, DEAEdextran, electroporation, protoplast fusion, and liposomes.Transplantation of normal genes into the affected tissues of a patientcan also be accomplished by transferring a normal NRG-2 gene into acultivatable cell type ex vivo, after which the cell (or itsdescendants) is injected into a targeted tissue. Another strategy forinhibiting NRG-2 function using gene therapy involves intracellularexpression of an anti-NRG-2 antibody or a portion of an NRG-2 antibody.For example, the gene (or gene fragment) encoding a monoclonal antibodythat specifically binds to NRG-2 and inhibits its biological activity isplaced under the transcriptional control of a tissue-specific generegulatory sequence. Another therapeutic approach involvesadministration of recombinant NRG-2 polypeptide, either directly to thesite of a potential or actual disease-affected tissue (for example, byinjection) or systemically (for example, by any conventional recombinantprotein administration technique). The dosage of systemically deliveredNRG-2 depends on a number of factors, including the size and health ofthe individual patient but, generally, between about 0.006 mg/kg toabout 0.6 mg/kg, inclusive, is administered per day to an adult in anypharmaceutically acceptable formulation. Dosages of NRG-2 delivered bylocal delivery may differ from systemic delivery, and can be determinedusing standard techniques known to those of ordinary skill in the art.

In a patient diagnosed as having a NRG-2 mutation or NRG-2-relateddisease, or as susceptible to NRG-2 mutations, aberrant NRG-2 expression(even if those mutations or expression patterns do not yet result inalterations in NRG-2 expression or biological activity), or to aNRG-2-related disease, any of the above-described therapies areadministered before the occurrence of the disease phenotype. Also,compounds shown to modulate NRG-2 expression or NRG-2 biologicalactivity are administered to patients diagnosed with potential or actualdiseases by any standard dosage and route of administration.Alternatively, gene therapy using an antisense NRG-2 mRNA expressionconstruct is undertaken to reverse or prevent the gene defect prior tothe development of the full course of the disease.

The therapeutic methods of the invention are, in some cases, targeted toprenatal treatment. For example, a fetus found to have a NRG-2 mutationis administered a gene therapy vector including a normal NRG-2 gene ornormal NRG-2 protein. Such treatment may be required only for a shortperiod of time, or may, in some form, be required throughout such apatient's lifetime. Any continued need for treatment, however, isdetermined using, for example, the diagnostic methods described above.Also as discussed above, NRG-2 abnormalities may be associated withdiseases in adults, and thus, adults are subject to the therapeuticmethods of the invention as well.

Identification of Molecules that Modulate NRG-2 Biological Activity orWhose Biological Activity is Modulated by NRG-2

Isolation of NRG-2 cDNAs (as described herein) also facilitates theidentification of molecules that increase or decrease NRG-2 biologicalactivity. Similarly, molecules whose activity is modulated by NRG-2biological activity can be identified. According to one approach,candidate molecules are added at varying concentrations to the culturemedium of cells expressing NRG-2 mRNA. NRG-2 biological activity is thenmeasured using standard techniques. The measurement of biologicalactivity can include, without limitation, the measurement of NRG-2protein and nucleic acid molecule levels, and NRG-2 phosphorylation.

If desired, the effect of candidate modulators on expression can also bemeasured at the level of NRG-2 protein production using the same generalapproach and standard immunological detection techniques, such aswestern blotting or immunoprecipitation with a NRG-2-specific antibody(see below).

A test compound that is screened in the methods described above can be achemical, be it naturally-occurring or artificially-derived. Suchcompounds can include, for example, polypeptides, synthesized organicmolecules, naturally occurring organic molecules, nucleic acidmolecules, and components thereof. Candidate NRG-2 modulators includepeptide as well as non-peptide molecules (e.g., peptide or non-peptidemolecules found, e.g., in a cell extract, mammalian serum, or growthmedium in which mammalian cells have been cultured).

Administration of NRG-2 Polypeptides, NRG-2 Nucleic Acid Molecules, andModulators of NRG-2 Synthesis or Function

A NRG-2 protein, nucleic acid molecule, modulator, neutralizing NRG-2antibody, or NRG-2-inhibiting compound (e.g., antisense NRG-2 or a NRG-2dominant negative mutant) is administered within apharmaceutically-acceptable diluent, carrier, or excipient, in unitdosage form to patients or experimental animals. Also, conventionalpharmaceutical practice is employed to provide suitable formulations orcompositions in which to administer such molecules or compounds topatients suffering from a NRG-2-related disease, such as demyelinatingdisorders of the peripheral and central nervous systems; neuropathies;neurodegenerative disorders; cardiomyopathies; loss of hearing, balanceor vision; pain; neurotrauma; cancer; sensorineural hearing loss orsensorineural balance loss from viral infection, aging, or antibiotics(e.g. aminoglycosides); retinopathy (e.g., hypertensive, diabetic,occlusive, macular degeneration, retinitis pigmentosa, optic neuropathy,injury); Guillaime Barre disease; stroke; or brain or spinal cordinjury. Administration can begin before or after the patient issymptomatic.

Any appropriate route of administration can be employed, for example,administration can be parenteral, intravenous, intra-arterial,subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraspinal, intracisternal,intraperitoneal, intranasal, inhalation to deep lung, aerosol, bysuppositories, oral, or topical (e.g. by applying an adhesive patchcarrying a formulation capable of crossing the dermis and entering thebloodstream). Preferably, the administration is local to the afflictedtissue, such as cardiac, lung, or nerve tissue. Therapeutic formulationscan be in the form of liquid solutions or suspensions; for oraladministration, formulations can be in the form of tablets or capsules;and for intranasal formulations, in the form of powders, nasal drops, oraerosols. Any of the above formulations may be a sustained-releaseformulation.

Methods that are well known in the art for making formulations arefound, for example, in Remington's Pharmaceutical Sciences, (18^(th)edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.Formulations for parenteral administration can, for example, containexcipients; sterile water; or saline; polyalkylene glycols, such aspolyethylene glycol; oils of vegetable origin; or hydrogenatednapthalenes. Sustained-release, biocompatible, biodegradable lactidepolymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers can be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for NRG-2 modulatory compounds include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation can contain excipients, forexample, lactose, or can be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate, and deoxycholate, or canbe oily solutions for administration in the form of nasal drops, or as agel.

Synthesis of NRG-2 Proteins, Polypeptides, and Polypeptide Fragments

Those skilled in the art of molecular biology will understand that awide variety of expression systems can be used to produce therecombinant NRG-2 proteins. The precise host cell used is not criticalto the invention. The NRG-2 proteins can be produced in a prokaryotichost (e.g., E. coli) or in a eukaryotic host (e.g., S. cerevisiae,insect cells such as Sf9 cells, or mammalian cells such as COS, NIH 3T3,CHO, or HeLa cells). These cells are commercially available from, forexample, the American Type Culture Collection, Rockville, Md. (see alsoAusubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, N.Y., 1998). The method of transformation and the choiceof expression vehicle (e.g., expression vector) will depend on the hostsystem selected. Transformation and transfection methods are described,e.g., in Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y., 1998, and expression vehicles can bechosen from those provided, e.g. in Pouwels et al., Cloning Vectors: ALaboratory Manual, 1985, Supp. 1987).

The characteristics of NRG-2 nucleic acid molecules are analyzed byintroducing such genes into various cell types or using in vitroextracellular systems. The function of NRG-2 proteins produced in suchcells or systems are then examined under different physiologicalconditions. Also, cell lines can be produced that over-express the NRG-2gene product, allowing purification of NRG-2 for biochemicalcharacterization, large-scale production, antibody production, andpatient therapy.

Use of NRG-2 Antibodies

Antibodies to NRG-2 proteins (for example, those described herein) areused to detect NRG-2 proteins or to inhibit the biological activities ofNRG-2 proteins. For example, a nucleic acid molecule encoding anantibody or portion of an antibody can be expressed within a cell toinhibit NRG-2 function. In addition, the antibodies can be coupled tocompounds, such as radionuclides and liposomes for diagnostic ortherapeutic uses. Antibodies that inhibit the activity of a NRG-2polypeptide can also be useful in preventing or slowing the developmentof a disease caused by inappropriate expression of a wild type or mutantNRG-2 gene.

Construction of Transgenic Animals and Knockout Animals

Characterization of NRG-2 genes provides information that allows NRG-2knockout animal models to be developed by homologous recombination.Preferably, a NRG-2 knockout animal is a mammal, most preferably amouse. Similarly, animal models of NRG-2 overproduction can be generatedby integrating one or more NRG-2 sequences into the genome of an animal,according to standard transgenic techniques. Moreover, the effect ofNRG-2 gene mutations (e.g., dominant gene mutations) can be studiedusing transgenic mice carrying mutated NRG-2 transgenes or byintroducing such mutations into the endogenous NRG-2 gene, usingstandard homologous recombination techniques.

A replacement-type targeting vector, which can be used to create aknockout model, can be constructed using an isogenic genomic clone, forexample, from a mouse strain such as 129/Sv (Stratagene Inc., LaJolla,Calif.). The targeting vector can be introduced into a suitably-derivedline of embryonic stem (ES) cells by electroporation to generate ES celllines that carry a profoundly truncated form of a NRG-2 gene. Togenerate chimeric founder mice, the targeted cell lines are injectedinto a mouse blastula-stage embryo. Heterozygous offspring can beinterbred to homozygosity. NRG-2 knockout mice provide a tool forstudying the role of NRG-2 in embryonic development and in disease.Moreover, such mice provide the means, in vivo, for testing therapeuticcompounds for amelioration of diseases or conditions involving aNRG-2-dependent or NRG-2-affected pathway.

The following Examples will assist those skilled in the art to betterunderstand the invention and its principles and advantages. It isintended that these Examples be illustrative of the invention and notlimit the scope thereof.

Example 1

Cloning of Human NRG-2 cDNA

A full-length cDNA encoding NRG-2α was identified from cerebellum.Multiple probes to various regions of NRG-2 coding sequences weredesigned based on rodent and human sequence data for cloning, mappingand sequence analysis. Prior to screening libraries, the specificity ofthe probes was confirmed by analyzing human cerebellar RNA using a 3′RACE (rapid amplification of cDNA ends) approach. Approximately 400,000cDNAs from two human cerebellum γt10 cDNA libraries (ClontechLaboratories, Palo Alto, Calif.; Catalog No. HL1128a) were screened withan oligonucleotide probe: 5′ GCA TCA ACC AGC TCT CCT GC 3′ (SEQ ID NO:5) from the EGFL domain of NRG-2. Twenty five hybridization signals weredetected; twenty of the phage clones corresponding to these signals werecloned and further analyzed by hybridization studies, physical mapping,and DNA sequencing. The results of these analyses were consistent withthe existence of multiple structural variants (isoforms) among the humanNRG-2 clones that were identified. Preliminary structural information onthe clones was obtained by filter hybridization to phage plaques andrestriction endonuclease analyses of the cDNA inserts. PCR studies,using internal primers, in pairs or in combination with flankingsequences, were used to obtain physical mapping data (see Table 1).

The primers used were as follows:

Primer 1471: (SEQ ID NO: 6) 5′ - GCA TCA ACC AGC TCT CCT GC - 3′Primer 1494: (SEQ ID NO: 7) 5′ - TGC GAA CTG CTG ACA CCT GT - 3′Primer 1527: (SEQ ID NO: 8) 5′ - CCA CCT TTT GAG CAA GTT CAG - 3′Primer 1528: (SEQ ID NO: 9) 5′ - GAG GTG GCT TAT GAG TTC TTC - 3′Primer 1531: (SEQ ID NO: 10) 5′ - GGC CAC CAC ACA GAC GAT G - 3′

First, the insert sizes, which ranged from 0.8 kb to 3.3 kb (averagesize was roughly 1.7 kb), were analyzed. NRG-2 transcripts contain anEGFL domain and cytoplasmic sequences that exhibit much of thestructural diversity of these polypeptides, and this specific internalregion was focused on next to map the clones by PCR analysis. Thisanalysis yielded four groups of products, and multiple clones wereidentified in each group. Therefore, the four groups (A-D) are likely torepresent the extent of structural diversity in this region among theNRG-2 gene products in human cerebellum. Four clones (group A) gave noproduct in this experiment. This result was consistent with the datafrom hybridization experiments, which had shown that these clones lackedthe sequence of the downstream primer (in the cytoplasmic domain). Inthe third experiment, the orientation of the clones was determined andthe distance from the EGFL domain to the ends of the clones wasestimated by using primers in the EGFL domain in combination withprimers from flanking sequences in the phage arms. These studies,therefore, enabled the segregation of the NRG-2 cDNAs into groups, andfacilitated identification of potential full-length cDNAs encodingsecretable isoforms of human NRG-2.

TABLE 1 Mapping human NRG-2 cerebellar cDNA clones Internal Largest 5′end to Group: clones Product¹ Clone: size² EGFL³ EGFL to 3′ end⁴ A: 1,4, 13, 15 None 13: 3300 1400 2300 B: 3, 6, 7, 9, 170 11: 1050 600 45011, 18, 20 C: 10, 12, 14, 260 14: 1500 850 650 16 D: 5, 8, 17, 19 650 8: 1600 800 800 PCR analyses of cDNA clones: products were sized on 6%polyacrylamide gels; the table showns sizes in base pairs. ¹Upstreamprimer 1471 from EGFL domain; downstream primer 1531 from cytoplasmicdomain. ²Primers 1527, 1528 from flanking sequences in λgt10. ³Upstreamprimer 1527 from flanking sequences in λgt10; downstream primer 1494from EGFL domain. ⁴Upstream primer 1471 from EGFL domain; downstreamprimer 1528 from flanking sequences in λgt10.

Example 2 Human NRG-2 DNA Sequence Analysis

To obtain a more complete picture of the different structures, DNAsequencing was undertaken on representative clones from each group usinga cycle sequencing protocol and the same primers used for the PCRanalysis described above. Comparison of the sequence contigs surroundingthe EGFL domain (from groups B-D) to each other and to rat and humanNRG-2 sequences led to several conclusions. First, the group B clonesmatched the NRG-2β cDNA structure. These sequences connected the EGFLdomain to the transmembrane and cytoplasmic domains, and thus encodedmembrane-attached NRG-2 protein. Second, all of the group C structurescontained both the α and the β sequences, and matched the structure ofthe NRG-2α cDNA. Therefore, group C clones should encode a secretedNRG-2 protein. Clone 14 appeared to be the best candidate for afull-length version of this structure. In group D, both α and βsequences were present, but they were not adjacent. A 450 bp sequenceintervening between these two known coding sequences was found, andimmediately adjacent to the regions identified as a and β sequences werecanonical splice junction donor (GT) and acceptor (AG) sequences. Thus,this structure probably represents a partially spliced transcript of theNRG-2 gene.

Given this information, it appeared that secretable forms of NRG-2 weremost likely be found in the clones of groups A and C. Clone 14 served asa suitable representative of group C. Two group A clones were advancedin parallel; clone 13 was selected because of the relatively largeinsert size and clone 15 was pursued because of the presence of sequence5′ of the EGFL domain that was detected in hybridization experiments.When sequences of clones 13, 14 and 15 were completed it became apparentthat none of them alone encoded a full length human NRG-2α. However,given the substantial overlap in the structure of these clones, it wasclear that portions of each could be spliced together to generate onefull length clone encoding NRG-2α. FIG. 1 shows a schematicrepresentation of the structure of these sequences; coding segments ofthe NRG-2 gene are shown in the shaded boxes, and the coding sequences(solid lines) that are present in described NRG-2α and NRG-2β isoformsare drawn above; isoforms of NRG-2 contain GGF2-like,immunoglobulin-like (Ig), EGF-like (EGFL), α, β, transmembrane (M), andcytoplasmic (cyto) domains; stop codons are indicated by (*); andputative intron sequences are represented by dashed lines. A uniqueBsrGI site (B) present in the α coding segment was used to construct afull-length human NRG-2α cDNA by connecting 5′ sequences of clone 15 tothe 3′ sequences of clone 14, and the sequence of the final constructwas determined. The major open reading frame of the NRG-2α cDNA (SEQ IDNO: 1) encodes a 330 amino acid protein (SEQ ID NO: 2).

Example 3

Cloning and Construction of Human NRG-2b cDNA

The human NRG-2β cDNA is constructed partly from the human NRG-2a cDNA(the vector and the 5′ 869 bp of sequence encoding the N-terminus ofhuman NRG-2, which is present in both the α and the β isoforms) andpartly from a phage clone (e.g., phage clone 11 was shown in mappingstudies to contain the β sequence—see Table 1, Example 1, and Example 2)containing a partial human cDNA encoding human NRG-2β (two 3′ fragments:one contains the β sequence and the other a stop codon).

The human NRG-2α cDNA (Example 2) can be digested with enzymes Not I andXba I (New England Biolabs, Beverly, Mass.) to generate a 5500 bp vectorand a 1555 bp insert containing the cDNA. Both fragments are recoveredfrom a gel of 1% agarose in TAE buffer using the QIAEX II Agarose GelExtraction kit and protocol (Qiagen, Inc., Valencia, Calif.). The insertfragment (1555 bp) is further digested using Drd I (New England Biolabs,Beverly, Mass.) to generate a 5′ 869 bp fragment and a 3′ fragment ofapproximately 700 bp. The 869 bp Not I-Drd I fragment is recovered froma gel of 2% agarose in TAE buffer using the QIAEX II Agarose GelExtraction kit and protocol (Qiagen, Inc., Valencia, Calif.). This 869bp fragment contains the initiator methionine and encodes the N-terminalportion of human NRG-2β. It is ligated into the 5550 bp vector alongwith two additional fragments, which are derived from cDNAs that havesequences of human NRG-2β as described below.

The major difference between the human NRG-2α and human NRG-2β sequencesreside between the single Drd I and Bsr DI sites. The α isoform containsa 77 bp coding segment that is spliced into the β isoform sequence. Toobtain the sequences encoding human NRG-2β, a 113 bp Drd I-BsrDIfragment, which is 77 bp shorter than the corresponding sequence ofhuman NRG-2α, is generated from phage clone 11 as follows. Primers(1551: 5′-GTG-AGC-ACC-ACC-CTG-TCA-TC-3′, SEQ ID NO: 11; 1546:5′-GAG-CTA-GTC-TAG-AGT-GGC-TTA-TGA-GTA-TTT-CTT-C-3′, SEQ ID NO: 12)flanking the Drd I and BsrDI sites are used to amplify the phage clone11 DNA template following methods recommended by the supplier of TaqPolymerase (Perkin Elmer/Roche, Branchburg, N.J.). The PCR product isprecipitated with ethanol, then digested sequentially using Drd I andBsrDI to produce a 113 bp fragment. Similarly, the 3′ fragment also isderived by PCR implication of the phage clone 11 template using primers1550 and 1546. Primer 1550(5′-CAG-CAG-TTC-GCA-ATG-GTC-AAC-TTC-TCC-TAA-GCA-CC-3′, SEQ ID NO: 13) ispositioned to cross over the BsrDl site and contains an insertion of asingle T that will mutate the target sequence5′-CAG-CAG-TTC-GCA-ATG-GTC-AAC-TTC-TCC-AAG-CAC-C-3′, SEQ ID NO: 14, to5′-CAG-CAG-TTC-GCA-ATG-GTC-AAC-TTC-TCC-TAA-GCA-CC-3′, SEQ ID NO: 15, andthus, convert a lysine codon to a TAA stop codon. Primer 1546 istargeted to the phage right arm cloning site and contains an Xba I site.Digestion of the product using BsrDI and Xba I generates a 425 bpfragment that becomes the 3′ end of the human NRG-2β cDNA. Both the 113bp and 425 bp fragments are recovered from 2% agarose gels.

The recovery of fragments is quantified by electrophoresis relative todouble stranded DNA markers of known length and quantity (e.g., phagelambda Hind III digest; New England Biolabs, Beverly, Mass.; pGEMmarkers, Promega, Madison, Wis.), and then each purified fragment isconverted into molar equivalents. The purified vector (100 ng) and thethree fragments are ligated together (T4 DNA ligase; New EnglandBiolabs, Beverly, Mass.) at equimolar ratios according to instructionsprovided by the supplier. The ligations are used to transform competentbacterial cells, such as E. coli XL1 Blue (Stratagene, La Jolla, Calif.)according to instructions provided by the supplier. Colonies containingthe vector are selected on the basis of resistance to 50 ug/mlampicillin, and the structure of the human NRG-2β cDNA is analyzed byPCR amplification and DNA sequencing of plasmid DNA. The major openreading frame of the human NRG-2β cDNA (SEQ ID NO: 3) encodes a proteinof 298 amino acids (SEQ ID NO: 4).

Example 4 Expression of Human NRG-2

A vector for transient and stable expression of human NRG-2 in mammaliancells was constructed. The pRc/CMV2 vector (Invitrogen V750-20; see FIG.2) was used to express human NRG-2. This 5.5 kb vector utilizes a CMVpromoter and a bovine growth hormone polyadenylation site to drive highlevel constitutive expression in both transient and stabletransfections. Neomycin selection (G418) can be used to select forstable transformants. The human NRG2-α cDNA sequence (SEQ ID NO: 1) wascloned directionally into the polylinker using the Hind III and Xba Isites. The cDNA insert of the final construct was sequenced on bothstrands. The human NRG-2 expression vectors were then expressed in CHOcells to provide a reliable source of recombinant human protein.

Both human NRG-2 α and β cDNAs were transiently transfected into CHO/Scells (Life Technologies, Inc., Rockville, Md.). Heterologous expressionof transfected genes was performed to ensure the proper functioning ofthe CHO/S cell system. Mock transfections were performed in parallel.Transfections were done in 100 mm dishes (in triplicate) by theLipofectamine™ 2000 method according to protocols supplied by themanufacturer (Life Technologies, Inc., Rockville, Md.). Cell lysates andconditioned media samples were collected 3 or 4 days post-transfection.To prepare lysates, cell monolayers were washed with PBS, scraped fromthe dishes, and lysed by three freeze-thaw cycles in 150 μl 0.25 M TrisHCl pH 8. Cell debris was pelleted and the supernatant recovered.Conditioned media samples were collected, then either analyzed directlyor concentrated and buffer-exchanged with 10 mM Tris HCl, pH 7.4 usingCentricon-10 units (Ambion). Secretion of biologically activerecombinant human NRG-2 gene product was demonstrated by stimulation ofSchwann cell proliferation following transient transfection of CHO/Scells and detection of NRG-2 bioactivity in the conditioned media ascompared to the cell lysate (Marchionni et al., Nature 362: 312-8,1993).

The recombinant human NRG-2 (rhNRG-2) proteins were efficientlyexpressed in the conditioned media of the transfected cells (FIGS. 3Aand 3B), indicating these proteins can be successfully secreted frommammalian cells. In FIG. 3A, the conditioned media, but not the celllysate from cells transfected with rhNRG-2α (and not frommock-transfected cells) expressed a specific immunoreactive band runningat ca. 56 kD. In FIG. 3B, both the α and β isoforms were secreted intothe conditioned media of transfected CHO/S cells (note the smallerrhNRG-2β protein (298 amino acids) ran faster (ca. 47 kD) than therhNRG-2α protein (331 amino acids), as expected).

After confirming the bioactivity of the expression construct intransient transfections, stable CHO/S cell lines were generated toexpress rhNRG-2β. The pRc/CMV2 vector contains a Neomycin resistancegene, so stably-transformed cells can be selected in media that containan effective concentration of G418. Following transfection of recipientCHO/S cells, well-isolated colonies that survived 11 days in theselective media were picked using cloning rings. Cell lines showing thehighest level expression of rhNRG-2β and G418 resistance were continuedfor further evaluation. Three useful properties of cell lines aresustained viability, adaptation to serum-free (or low serum) media, andexpression level of recombinant protein. Thus, several of the lines wereexpanded in parallel and tested for adaptation to serum-free growthconditions and expression of rhNRG-2β by western blot. Western blotanalysis showed that Dulbecco's modified essential medium supplementwith 2% fetal calf serum provided for optimum expression of rhNRG-2β inthese experiments. Bioactivity was assayed on expressed material fromthe leading candidate lines. Two isolates were cloned by limitingdilution, and a single isolated cell line was used for further studies.

In addition to generating stable CHO/S cell lines expressing NRG-2proteins, a strategy relying on the co-amplification of integratedcopies of rhNRG-2β expression constructs and a transfected dihydrofolatereductase (dhfr) gene was developed. Mammalian expression vectors wereconstructed in pcDNA3.1 (Invitrogen) and pMACSK^(k).II (Miltenyi BiotecLtd.) under the control of the CMV and SV40 promoters, respectively.These vectors were co-transfected along a dhfr expression vector intoCHO-dhfr cells and thirty colonies resistant to G418 were selected,grown up and expression levels were analyzed by western blot (FIG. 6)and RT/PCR. FIG. 6 Lane 1 shows rhNRG-2β control sample, Lane 2 showsCHO SD (US) rhNRG2β 48 hr supernatant, Lanes 3-5 show 24-72 hrs CloneT3B2, Lanes 6-8 show 24-72 hrs Clone T3B1, Lane 9 shows molecular weightmarkers (Invitrogen cat #LC5925), Lanes 10-12 show 24-72 hrs Clone T3A6,and Lanes 13-15 show 24-72 hrs Clone T3A5. The T3B2, T3B1, T3A5 cloneswere selected for dilution cloning and for amplification/selection. Geneco-amplification is induced by step-wise increase in methotrexateconcentrations and clones are monitored for increases in yield ofsecreted rhNRG2β.

Example 5 Generation and Testing of Antisera to Detect Expressed NRG-2Protein

A polyclonal antiserum that specifically detects expressed NRG-2 proteinwas generated as follows. Peptides were designed from the deduced humanand rat NRG-2 sequences to generate rabbit polyclonal antisera to beused to monitor NRG-2 levels in expression and purification samples.

The peptides used were as follows:

K71983M: (SEQ ID NO: 16) APLERNQRYIFFLEPTEQPLVFK K71984M:(SEQ ID NO: 17) NSRLQFNKVKVEDAGEY K71985K: (SEQ ID NO: 18)NGGVCYYIEGINQLS

One of these peptides (K71984M), derived from the Ig domain sequence,which is identical in the deduced rat and human NRG-2 sequences,produced useful sera for western blotting recombinant rat NRG-2β inconditioned media from transfected CHO cells (see FIG. 3C) and did notcross-react with rhGGF2. These NRG-2 antisera were purified against theimmobilized peptide.

FIG. 3C shows a western blot analysis of conditioned media from CHOcells expressing rat NRG-2β. The lanes contain either 20 μl of 15-foldconcentrated conditioned medium from CHO cells expressing rrNRG-2β(left) or 10 ng rhGGF2 (right). Anti-NRG-2 serum was used at 1 g/ml andspecifically detected rrNRG-2β, at 46 kD, but not rhGGF2, which runs at80 kD.

Furthermore, analysis of culture media from transiently transfectedmonolayers of CHO/S cells, using the expression plasmids rhNRG-2α andrhNRG-2β and a rabbit polyclonal antibody raised against peptideK71984M, indicated that both rhNRG-2α and rhNRG-2β were expressed, andmigrated at approximately 55 kD and 47 kD, respectively (FIG. 5).

Example 6

Bioassay for Assessment of Biological Activity of Expressed rhNRG-2α andrhNRG-2

A bioassay for detection of biologically-active rhNRG-2α was developed.Neuregulin signalling occurs through erbB receptor tyrosine kinasesbelonging to the EGF receptor family. NRG ligand binding and receptoractivation can be detected by western blotting of treated cell lysatesusing antisera directed against phosphorylated tyrosine residues. Thisassay is used to study the interactions of NRG-2 proteins and erbBreceptors in a variety of cell types including, but not limited to,Schwann cells, oligodendrocyte progenitors, skeletal myotubes,cardiomyocytes, and human tumor cell lines from breast and prostateadenocarcinomas. Biologically-active NRG-2 (e.g., conditioned mediumfrom CHO cells expressing recombinant rat NRG-2β) can be detected usingthis assay on the human breast adenocarcinoma cell line MCF-7. Theresults of an experiment testing rat NRG-2β (rrNRG-2β) and rhGGF2 onMCF-7 cells by a receptor phosphotyrosine western blot is shown in FIG.4A. MCF-7 cells were cultured in 24 well plates (2×10⁵ cell/well) andtreated for 15 minutes in DMEM-0.1% FCS containing 10 ng/ml rhGGF2, orvarious dilutions of medium conditioned by CHO-cells expressingrhNRG-2β. Following the treatment, the media were removed and the cellswere washed once, then the cultures were lysed, and samples wereanalyzed by western blotting (Canoll et al, Neuron 17, 229-243, 1996).Phosphorylated ErbB receptors are detected with the RC20Bphosphotyrosine antibody (Transduction Laboratories, Lexington, Ky.). Apositive control sample for this analysis is a lysate of A431 cellstreated with EGF (left-most lane; FIG. 4A). When neither rhGGF2 norrrNRG-2β were added to the growth media, there were no detectableproteins phosphorylated on tyrosine. However, addition of variousconcentrations of NRG-2β showed a dose dependent increase inphosphorylation at 185 kd. This band matched the expected position ofthe ErbB2 and ErbB3 receptors, which were also phosphorylated inresponse to treatment with rhGGF2 (10 ng/ml). Therefore, this bioassayprovides a reliable method to verify the bioactivity of expressed andpurified rhNRG-2α and rhNRG-2β. This assay, when applied to purifiedrecombinant protein, enables quantification of the bioactivity of NRG-2in dose response curves that provide comparable data to DNA synthesisassays. Receptor tyrosine kinase bioassays on MCF-7 cells treated withconditioned media from CHO/S cell transient transfections or withpurified recombinant NRG proteins are also shown in FIG. 4B.

Example 7

Purification of Milligram Quantities of rhNRG-2α or β

Conditioned medium harvested from a producer cell line (expressingrhNRG-2α) is adjusted to pH 6.0 with acetic acid and loaded directlyonto an S-sepharose column equilibrated with sodium acetate (pH 6.0).Bound material is eluted with 1M NaCl in acetate buffer, equilibrated inammonium sulfate buffer and passed over a hydrophobic interaction column(Butyl Sepharose FF) in the same buffer. Bound material is eluted withlow salt (800 mM ammonium sulfate) buffer and the rhNRG-2α peakcollected. Collected material is buffer exchanged and concentrated to 1mg/ml in formulation buffer (100 mM arginine, 100 mM Sodium Sulphate, 20mM NaAc, 1% mannitol pH 6-7) using an Amicon spiral cartridge. Anoptional, final purification step is a Sephacryl 200 HR column andeluted rhNRG-2α peak is formulated in formulation buffer. An alternativeapproach is to follow the purification scheme relying on heparinaffinity, Cu-chelate and C4-reversed phase chromatographies (Higashiyamaet al., J. Biochem. 122: 675-680, 1997).

Proteins fractions from chromatographic columns are monitored by westernblotting (e.g., see FIG. 3A-C) to identify the peak of secreted rhNRG-2αor β. Peak fractions and final preparations are analyzed by receptorphosphorylation on MCF-7 cells (see FIG. 4A-B). Purity is assessed bygel electrophoresis (coommassie blue staining) and by analytical HPLC(Vydac C8 column run in a gradient of acetonitrile in 0.1%trifluoracetic acid). Protein concentrations are determined by thebicinchoninic acid (BCA) assay (Pierce) with bovine serum albumin usedas a standard.

Another general plan for purification involves capture by conventionalchromatography on cation exchange followed by resolution fromcontaminating proteins through one or more steps, for example, byutilizing carboxymethyl Sepharose chromatography followed by reversephase HPLC.

Briefly, carboxymethyl sepharose (fast flow) columns of varying sizeswere equilibrated with 200 mM NaCl 10 mM Tris pH 7.4, then conditionedmedia samples were loaded, and the column was washed with approximately3 volumes of 200 mM NaCl, 10 mM Tris pH 7.4 (until the absorbance hadreached baseline). Bound protein was eluted with 500 mM NaCl, 10 Mm Trisat pH 7.4. This elution was followed by a high salt wash (1M NaCl, 10 mMTris pH 7.4) for 3 column volumes. Fractions were collected and analyzedby western blot and gold or coommassie blue stained protein gels (4-20%acrylamide Tris-glycine-SDS). Depending on the column scale and thequantity of protein loaded captured, rhNRG-2β represented from 10-70% ofthe protein eluted in 0.5 M NaCl from the column. No detectable rhNRG-2βwas detected in the flowthrough, nor in the 0.2 M NaCl or 1M NaClfractions, provided that the column was not overloaded. Significantimprovements in recovery were obtained (>90%) by including proteaseinhibitors and running the column in the cold. The scale of capturechromatography was increased, beginning with 10 ml columns, through 40ml, 100 ml, and 200 ml columns. The overall results were consistent bothin terms of recovery and purification, indicating that the scale of thisstep can be adjusted to suit the volume of starting material available.

The purification method was further developed with reverse phase HPLCusing a C4 column (Vydac 214 TP 1010, 1 cm×25 cm column) operated on aBiocad Perfusion Chromatography Workstation. A series of pilot runs wereperformed on pooled fractions from several carboxymethyl sepharosecolumns that contained rhNRG-2β in 10 mM TrisHCl pH 7.4, 0.5 M NaCl. Thecolumn was operated at a flow rate of 1 ml/min and was equilibrated in0.2% TFA. After injecting the sample, a 10 min. column wash in 0.2% TFAwas followed with a 30 min. linear ramp up to 90% acetonitrile, 0.2% TFAand a final 10 min. wash step in 90% acetonitrile, 0.2% TFA was used tocomplete the method. Fractions were analyzed by western blot. Only thefractions that contained very pure rhNRG-2β were included in the finalpool. As assessed by coommassie blue staining of the gel shown in FIG.7A, the preparation of rhNRG-2β was approximately 92% pure. However,approximately 60-70% of the immunoreactivity detected across the HPLCchromatograph was not included in the rhNRG-2β pool. Therefore, although90% purification has been achieved in 2 steps, a third step may beperformed to enable more complete recovery of rhNRG-2β. This third stepmay include heparin sepharose and/or several modifications of thereverse phase HPLC step (e.g., variations in solvents).

Gold staining provides another sensitive method for detectingcontaminating proteins in protein preparations, and this stain readilydetects nanogram quantities of protein. To visualize the purificationprocess and to further analyze the purity of rhNRG-2β samples fromdifferent stages of purification were compared (FIG. 8). Samples wererun on 4%-20% SDS PAGE (Novex, cat #EC 6025) gels in reduced conditions.The gels were transferred onto PVDF membrane, and were stained for totalprotein with Gold Stain (Amersham, cat #RPN490). To prevent overloadingin the lane, the starting material (serum-free conditioned media) wasloaded at 1% of the relative amount of the purification samples. Thecentral observation from this analysis was that very significantpurification had been achieved in two steps.

The tyrosine phosphorylation assay performed on the MCF-7 cell line(human mammary adenocarcinoma) was used to measure the bioactivity ofpurified NRG-2 samples. Briefly, cultures were challenged with testsamples (dilutions of purification samples in medium containing 0.1%FCS) for 15 min at 37 C, then the media was aspirated, and 50 μl 2×sample buffer containing DTT and 1 mM sodium orthovanadate was added.Samples were then prepared for electrophoresis and Western blotting. Thecontrol sample, lysate from A431 cells treated with epidermal growthfactor, was provided by the vendor (Transduction Laboratories,Lexington, Ky.). In addition to monitoring rhNRG-2β production, theactivity of HPLC purified samples of rhNRG-2b diluted in vehiclescompatible with purification procedures such as 50% acetonitrile (AN) orPBS was examined (FIG. 9). At the concentrations used in thisexperiment, AN did not dramatically interfere with NRG signaling.

Example 8 NRG-2 Activities on Oligodendrocyte Progenitors

Evaluation of rhNRG-2α and rhNRG-2β effects on proliferation andsurvival of cultured oligodendrocyte progenitors is performed, usingrhGGF2 for comparison. Oligodendrocyte progenitors are generated from 2day old rat according to the method of McCarthy and DeVellis (J. CellBiol. 85: 890-902, 1980), and the cells are cultured in N2 defined mediacontaining 0.5% FBS (DM+) for one to three days to enrich for cells inthe oligodendrocyte lineage. Purity of the cultures is established byimmunofluorescence analysis using a series of antibodies directedagainst GFAP, a marker for astrocytes; OX42 monoclonal, a marker formicroglia (Harlan Bioproducts for Science); anti-A2B5 monoclonal(Boehringer Mannheim), a marker for 0-2A progenitors; O4 and O1, whichrecognizes early and mature oligodendrocytes respectively (Sommer etal., Dev. Biol 83: 311-327, 1980); RPTP-β (gift of J. Schlessinger, NYUMed. Ctr) and nestin antibodies (Developmental Studies Hybridoma Bank)which preferentially recognize early cells in the oligodendrocytelineage (Canoll et al., Neuron 17, 229-243, 1996; Gallo et al., J.Neurosci. 15: 394-406, 1995).

To determine the percentage of cells synthesizing DNA in response torhNRG-2α, rhNRG-2β, or rhGGF2 cultures are treated for 16 h and for thefinal 4 h in the presence of 10 μM bromodeoxyuridine (BrdU; Sigma).BrdU-labelled cells are detected using fluoroscein-conjugated anti-BrdUimmunodetection kit (Boehringer Mannheim). The labeling index,corresponding to the ratio of BrdU+ cells to total cells, is determinedfrom photomicrographs of individual fields of BrdU labeled and Hoechststained nuclei. To determine the labeling index at specific stages ofdifferentiation, BrdU staining is combined with analysis of 04, 01 andGFAP immunofluorescence.

To assess the effect NRG-2 on cell survival, cells growing in B104conditioned medium are changed to DM+ media for three days. They arethen switched to either N2 media or DMEM with or without rhGGF2,rhNRG-2α, or rhNRG-2β for 12 or 24 hours and stained with the Live/Deadstaining kit (Molecular Probes, Inc) for 15 min following themanufacturer's instructions. Morphologic criteria to quantify celldeath, i.e. monitoring pyknotic cells under phase microscopy and the MTTassay (Sigma), are used in separate experiments.

Example 9 NRG-2 Activities on Olfactory Bulb Ensheathing Cells

The rat olfactory bulb is an exceptional CNS tissue. Unlike other areasof the brain, growing axons are able to enter the olfactory bulb andextend within this CNS environment throughout adult life. The glialcells of the olfactory system, known as olfactory bulb ensheathing cells(OBECs), may have an important role in CNS neural regeneration (Li etal, J. Neurosci. 18: 10514-10524, 1998). OBECs are unusual glial cellspossessing properties of both astrocytes and Schwann cells, and may beuseful cells to aid in spinal cord regeneration. OBECs expressfunctional NRG receptors erbB2 and erbB4 (Pollock et al. Eur. J.Neurosci. 11: 769-780, 1999). Furthermore, high levels of NRG-2polypeptides are expressed in the olfactory bulb. Accordingly, theseOBECs are ideal candidates for comparing the bioactivities of NRG-1 toNRG-2 gene products.

OBECs are purified from postnatal day 7 rats by fluorescence activatedcell sorting using the 04 antibody (Barnett, In: Culture of AnimalCells, I. R. Freshney, 3rd Edition. pp 337-341. Wiley-Liss, New York,N.Y., 1993; Barnett et al., Dev Biol. 155: 337-350, 1993). Aftersorting, cell suspensions are plated onto coverslips and incubated inDMEM-BS containing 10% astrocyte conditioned medium (ACM) overnight at37° C. (to promote cell survival) before treatment with either growthfactors or ACM. Mitogenic activity is assayed by incorporation of BrdUinto dividing cells, and cell survival and apoptosis assays are done asdescribed (Pollock et al. Eur. J. Neurosci. 11: 769-780, 1999).

Example 10 NRG-2 Activities on Mid-Brain Dopaminergic Neurons.

The NRG receptor erbB4 is expressed in midbrain dopaminergic neurons ofthe rat, mouse, and monkey. Delivery of recombinant human NRG proteinsto the striatum is useful in the treatment of Parkinson's disease.Studies using the exemplary proteins, rhNRG-2α, rhNRG-2β, and rhGGF2 areundertaken to further investigate the response of the dopaminergicnigrastriatal system to NRGs. The two NRG proteins are compared forsurvival-promoting activity (i.e. protection from cell death induced byagents that induce oxidative stress) on dopaminergic neurons (forexample, from both fetal rodents and human neuroblastoma cells lines,e.g., SKNNC) in vitro. Cells pre-treated with varying concentrations ofrhNRG-2α, rhNRG-2β, or rhGGF2 are challenged with a twenty four hourtreatment of 1 μM metadione or 100 mM diethyldithiocarbamate to induceoxidative stress, and cell death is quantified by standard methods. Anin vivo model of dopamine release and electrochemical and behavioralassessments of dopaminergic function in rats can also be used.

NRG proteins were tested for survival promoting activity on ratdopaminergic neurons in vitro. Specifically, it was determined if NRGproteins were neuroprotective for dopaminergic neurons that werechallenged in culture with 6-hydroxydopamine (6-OHDA). Cells pre-treatedwith rhNRG-2β or rhGGF2 and untreated control cultures were exposed to50 μM 6-OHDA for 24 h, then cultures were stained for tyrosinehydroxylase (TH) and examined by light microscopy (FIGS. 10A-B). FIGS.10A-B show primary mesencephalic cultures immunostained for tyrosinehydroxylase (TH) on day in vitro (DIV) 7. The top panel shows a culturetreated with 100 nanograms rhNRG-2β daily starting at DIV 0 and endingat DIV 3. On DIV 4, the culture was treated with 50 μM 6-OHDA. Thebottom panel shows a culture that received no pretreatment, but wastreated with 50 μM 6-OHDA on DIV 4. Both cultures were analyzed for THimmunoreactivity on DIV 7. Calibration bar equals 50 microns and appliesto both top and bottom panels. Similar staining patterns were observedthroughout each of the cultures. The density, number, and length ofneurites of TH-positive neurons were reduced by 6-OHDA treatment in theculture receiving no pretreatment. In contrast, the culture pretreatedwith rhNRG-2β shows normal morphological development, which iscomparable to the results observed with rhGGF2. This result has beenreplicated in several culture experiments. The results indicate thatrhNRG-2β has beneficial effects in vivo, which can be tested, forexample, in an animal model of Parkinson's disease.

Example 11 Neuronal Development and Migration in the Cerebellum

Isoforms of NRG-1, NRG-2, and the erbB4 receptor are expressed at highlevels in the cerebellum (Chen et al., J Comp Neurol 349: 389-400, 1994;Chang et al., Nature 387: 509-512, 1997; Lai et al., Neuron 6: 691-704,1991). RhNRG-2α, rhNRG-2β, and rhGGF2 can be evaluated in cell cultureassays of migration and neurogenesis in the cerebellum. RhNRG-2α,rhNRG-2β, and rhGGF2 are compared with respect to their effect on therate of migration of cerebellar granule neurons on a glial cellsubstrate. Imprint cultures of postnatal day 5 rat cerebellum containingintact Bergmann glia with migrating neurons attached to them are made asdescribed (Anton et al., J. Neurosci. 16: 2283-2293, 1996). Neuronalmigration is monitored using a Zeiss axiovert 135 microscope equippedwith a Zeiss W63 objective, with images recorded onto an optical disk.Changes in the rate and pattern of neuronal migration, neuron-glialinteractions, and morphology are monitored in response to rhNRG-2α,rhNRG-2β, and rhGGF2.

Effects on cerebellar granule neurogenesis are studied in dissociatedcultures of postnatal rat cerebellar granular neurons. Dividing neuralprecursors are purified from postnatal day 5 cerebellum by Percolldensity gradient centrifugation and placed into dissociated cellculture. Cultures are then treated with 10 μM BrdUrd (to label dividingcells) and with varying concentrations of rhNRG-2α, rhNRG-2β, andrhGGF2. After two to seven additional days in culture, differentiationinto neuronal and glial cell lineages is assayed by immunostaining usingcell-type specific markers, such as GFAP (glial) and TUJ1 (neuronal).For each culture condition, the total number of cells, the BrdU-labelledcells, and the cells identified with each marker are enumerated. Cellsthat entered a particular cell lineage since exposure to these growthfactors are identified as those labelled with BrdU plus one of themarkers. The percentage of BrdU-labelled cells stained with each markerthus provides a measure of the effects of each growth factor on thegenesis and survival of neurons and glia. Analysis of total number ofcells at various time points and the number of apoptotic cells underdifferent conditions are used to evaluate any potential effect ofrhNRG-2α, rhNRG-2β, and rhGGF2 on selective survival of neuralprecursors or their neuronal or glial derivatives.

Isoforms of NRG-1, NRG-2 and the erbB4 receptor are expressed at highlevels in the cerebellum, thus making in vitro studies on neural cellsof the cerebellum an important component of these studies. Both rhNRG-2βand rhGGF2 were evaluated in cell culture assays of migration andneurogenesis in the cerebellum. Imprint cultures of postnatal day 5 ratcerebellum containing intact Bergmann glia with migrating neuronsattached to them were made and analyzed. Neuronal migration wasmonitored using a Zeiss axiovert 135 microscope equipped with a ZeissW63 objective, with images recorded onto an optical disk. Changes in therate and pattern of neuronal migration, neuron-glial interactions, andmorphology were monitored in response to rhnrg-2β and rhGGF2 (FIG.12A-B). The rate of neuronal migration of cerebellar granule neurons wasmeasured before and after exposure to rhNRG2β (100 ng/ml), rhGGF2 (50ng/ml), or unsupplemented control media. FIG. 12A shows neuronsmigrating on a glial cell process monitored prior to (panels to the leftof black arrow) and after (panels to the right of black arrow) additionof growth factors (shown here is rhGGF2). Time elapsed between eachpanel is one hour. FIG. 12B shows that exposure to rhGGF2 promoted therate of migration of neurons by 45±2.1%. In contrast, neither controlmedium nor rhNRG-2β altered the rate of migration. The asteriskindicates significance, P<0.05. Data shown are mean±SEM (n>16 for eachgroup). Therefore, in contrast to the observed increase in the rate ofneuronal migration promoted by rhGGF2, rhNRG-2β had no apparent effect.However, when cerebellar neural progenitors were studied in dissociatedculture, rhNRG-2β promoted external granule (EGL) neuron proliferationand/or survival (FIG. 11A-B). External granule layer (EGL) cells weredissociated and cultured in neurobasal (NB)/N2 medium or in NB/N2 mediumsupplemented with 100 ng/ml rhNRG-2β for 5 days. 10 μM BrdU was added toall cultures from the beginning. The cells then were fixed and probedwith a polyclonal neuron-specific antibody (Tuj-1; Babco) and withanti-BrdU monoclonal antibodies. Cells that were labeled with Tuj-1alone (i.e., neurons; asterisk[A]), BrdU+Tuj-1 (i.e., neurons generatedfrom dividing neuroblasts in culture; arrow [A]), and BrdU alone (i.e.,non-neural cells; arrowhead[A]) were counted. Compared to the control,more neurons (arrow, [A]) were found to have incorporated BrdU (orange)in their nuclei when cultured in medium containing rhNRG-2β. BrdUimmunoreactivity was detected with anti-mouse conjugated to Cy3 (Red).Tuj-1 immunoreactivity was detected with anti-rabbit conjugated to FITC.Numbers of Tuj-1 and BrdU positive cells were counted. Cell counts fromthe rhNRG-2β group was normalized to that from control group to obtainfold basal change in the number of BrdU positive neurons. This resultsuggests that rhNRG-2β promotes EGL cell proliferation or the selectivesurvival of newly generated cerebellar granule neurons. These datatherefore exemplify a bioactivity on neural cells that is more generallyapplicable to neuronal populations that express erbB4.

Example 12 NRG-2 Activities on Ventricular Cardiomyocytes.

To examine the role of NRG ligands and their receptors in developing andpostnatal myocardium, the ability of NRG-2 proteins to promoteproliferation, survival and growth of isolated neonatal and adult ratcardiac myocytes was studied. All three of the known receptors forneuregulins, erbB2, erbB3, and erbB4, are expressed in the developingheart at E14, after which erbB3 expression rapidly declines while erbB2and erbB4 expression persists in ventricular myocytes into adulthood.The in vitro activities of rhNRG-2α and rhNRG-2β on cardiomyocytes areevaluated as compared to rhGGF2. Specifically, the two growth factorsare compared for effects on cardiomyocyte survival, hypertrophy, andcontractile protein expression as described below. Neonatal ratventricular myocyte (NRVM) primary cultures are prepared as describedpreviously (Springhorn et al., J. Biol. Chem. 267: 14360-14365, 1992).To selectively enrich for myocytes, dissociated cells are centrifugedtwice at 500 rpm for 5 min, preplated twice for 75 min, and finallyplated at low density (0.7-1×104 cells/cm2) in DME medium supplementedwith 7% FBS. Cytosine arabinoside (AraC; 10M; Sigma) is added during thefirst 24-48 h to prevent proliferation of non-myocytes. Unless otherwisestated, all experiments are performed 36-48 h after changing to aserum-free medium, DME plus ITS (Sigma). Using this method, primarycultures with >95% myocytes are routinely obtained, as assessed bymicroscopic observation of spontaneous contraction and byimmunofluorescence staining with a monoclonal anti-cardiac myosin heavychain antibody (anti-MHC; Biogenesis, Sandown, N.H.).

Isolation and preparation of adult rat ventricular myocyte (ARVM)primary cultures is carried out using techniques previously described(Berger et al., Am. J. Physiol. 266: H341-H349, 1994). Rod-shapedcardiac myocytes are plated in culture medium on laminin- (10 (g/ml)precoated dishes for 60 min, followed by one change of medium to removeloosely attached cells. The contamination of ARVM primary cultures bynon-myocytes is determined by counting with a haemocytometer and istypically less than 5%. All ARVM primary cultures are maintained in adefined medium termed “ACCITT” (Ellingsen et al., Am. J. Physiol. 265:H747-H754, 1993) composed of DME, supplemented with 2 mg/ml BSA, 2 mML-carnitine, 5 mM creatine, 5 mM taurine, 0.1 (M insulin, and 10 nMtriiodothyronine with 100 IU/ml penicillin and 100 (g/ml streptomycin.In experimental protocols designed to examine myocyte survival and/orapoptosis, insulin is omitted from the defined medium, which istherefore termed “ACCTT”.

Measurements of rates of protein synthesis ([3H]leucine uptake) are usedto monitor growth factor effects on cardiomyocyte hypertrophy. For theseexperiments, 10 (M cytosine arabinoside is added to the culture medium.Cells are grown in serum-free medium for 36 to 48 h and then stimulatedwith different doses of rhNRG-2α, rhNRG-2β, or rhGGF2. After 40 h,[3H]leucine (5 (Ci/ml) is added for 8 hours, and cells washed with PBSand harvested with 10% TCA. TCA-precipitable radioactivity is determinedby scintillation counting.

Immunocytochemistry is used to examine changes in myocyte phenotype withrhNRG-2α, rhNRG-2β, or rhGGF2. For example, following treatment withgrowth factors, cells are fixed in 4% (w/v) paraformaldehyde for 30 minat room temperature, rinsed with PBS, permeabilized with 0.1% TritonX-100 for 15 min, and then incubated with 1% FBS for another 15 min,followed by incubation with anti-myosin heavy chain (1:300) andvisualized with TRITC-conjugated (NRVM) or FITC-conjugated (ARVM) secondantibody. ARVM are examined using a MRC 600 confocal microscope with aKr/Ar laser.

The in vitro activities of rhNRG-2α and rhNRG-2β on cardiomyocytes ascompared to rhGGF2 was evaluated. Studies on cellular hypertrophy (asmonitored by measuring protein synthesis) and activation of signallingpathways including p42/44 MAPK and Akt were performed (FIG. 13 and FIG.14). Neonatal rat ventricular myocytes isolated from 1 day old neontalrat ventricle were plated in 24 well tissue culture plates with ˜80,000cells/well for 24 hrs in 10% FCS, then serum starved overnight. Cellswere treated with recombinant neuregulins in the presence of ³H-leucinefor 24 hrs. Cellular protein was precipitated with 5% TCA and lysed with0.4N NaOH. ³H-leucine incorporation was measured with a scintillationcounter, and presented as the average of 4 wells treated identicallydivided by the average counts in untreated cells. Neonatal ratventricular myocytes were plated in p100s˜2-3 million cells/plate for 24hrs in 10% FCS, then serum starved 24 hrs. Cells were treated withrecombinant neuregulins for 10 min, then lysed with buffer containingprotease and phosphatase inhibitors (New England Biolabs). Samplesrepresenting 70 g protein were run on 10% gel (BioRad), and transferredto PVDF membrane for detection of phosphorylated Erk or Akt using NewEngland Biolabs phospho-specific antibodies. Both rhGGF2 and rhNRG-2βincreased protein synthesis by approximately 40% at all concentrationsexamined. However, rhNRG-2α had no effect on protein synthesis over theconcentration tested. The blot shown (FIG. 13) is representative of 2separate experiments.

These results indicate that NR2 signalling may act to promote theproliferation, survival, and growth of cardiac myocytes, both during andfollowing myocardial trabeculation. Moreover, the persistence of NRGreceptors in the postnatal and adult heart suggests a continuing rolefor neuregulins in the myocardial adaption to physiologic stress orinjury.

Example 13 Cell Survival Assay and Detection of Apoptosis

Cell viability is determined by the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT,Sigma) cell respiration assay. Primary cultures of NRVM after 2 days inserum-free medium are stimulated with different concentrations ofrhNRG-2α, rhNRG-2β, or rhGGF2 for either 4 or 6 days. ARVM aremaintained in ACCTT medium or ACCTT medium plus different concentrationsof rhNRG-2α, rhNRG-2β, or rhGGF2 for 6 days. MTT is then incubated withthe cells for 3 h at 37° C. Living cells transform the tetrazolium ringinto dark blue formazan crystals that can be quantified by reading theoptical density at 570 nm after cell lysis with dimethylsulfoxide.

Apoptosis is detected in neonatal and adult myocytes using the terminaldeoxynucleotidyltransferase (TdT)-mediated dUTP nick end-labeling(TUNEL) assay. 3′-end labelling of DNA with fluorescein-conjugated dUTPis done using an in situ cell death detection kit (Boehringer Mannheim)following the manufacturer's instructions. Cells are counterstained withan anti-MHC antibody as described above, and the nuclei are also stainedwith Hoescht 33258 (10 (M, Sigma) for 5 min. More than 500 myocytes arecounted in each coverslip and the percentage of TUNEL-positive myocytesis calculated.

OTHER EMBODIMENTS

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of theappended claims.

What is claimed is: 1-69. (canceled)
 70. A composition comprising arecombinant NRG-2 polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 4 for the treatment or prophylaxis of apathophysiological condition of the nervous system in a subject, andwherein said NRG-2 polypeptide is sufficient to induce myelination of aneuronal cell by a glial cell.
 71. The composition according to claim70, wherein said pathophysiological condition is selected from the groupconsisting of multiple sclerosis, amyotrophic lateral sclerosis,Parkinson's Disease, and spinal cord injury.
 72. The compositionaccording to claim 70, wherein said pathophysiological condition ismultiple sclerosis.
 73. The composition according to claim 70, whereinsaid pathophysiological condition is amyotrophic lateral sclerosis. 74.The composition according to claim 70, wherein said pathophysiologicalcondition is Parkinson's Disease.
 75. The composition according to claim70, wherein said pathophysiological condition is spinal cord injury. 76.The composition according to claim 70, wherein said glial cell is aSchwann cell or an oligodendrocyte.
 77. The composition according toclaim 70, wherein said subject is a human.
 78. A kit for the analysis ofa NRG-2 polypeptide of a test subject, said kit comprising the antibodythat specifically binds to a NRG-2 polypeptide comprising the amino acidsequence set forth in SEQ ID NOs: 2 or 4.